Bifunctional starch nanoparticles for achieving recyclable interfacial biocatalysis within a narrow pH range

Pickering interfacial biocatalysis (PIB) often suffers from the mismatch between the neutral pH required for enzyme activity and highly acidic conditions needed for emulsion destabilization during catalyst recycling. To address this, bifunctional starch nanoparticles were designed through sequential TEMPO-mediated oxidation and free-radical graft copolymerization with pH-responsive poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). The resulting nanoparticles, when optimized with a balanced carboxyl-to-amine ratio, incorporated both carboxylate anions and protonated tertiary amines, enabling fully reversible, pH-triggered emulsion breakdown within a narrow, enzyme-compatible window (pH 6-8). We mechanistically explained this behavior by interfacial analysis (zeta potential, contact angle, interfacial tension), which revealed that enhanced sensitivity stemmed from cooperative ionization of the bifunctional groups, together with ionic and buffering effects from phosphate-mediated pH adjustment. The bifunctional nanoparticles were used to immobilize Candida antarctica lipase B (CalB) for the esterification of glycerol and butyric acid, achieving 97% conversion in the resulting Pickering emulsion microreactor. The catalyst could be recovered and reused over eight cycles via simple pH switching between 8 and 6, retaining >80% initial activity without the enzyme deactivation associated with conventional low-pH demulsification. This work provides a sustainable, starch-based, and operationally simple strategy for advancing recyclable biocatalysis toward greener and more economical processes.